An ALPS-like patient without an identified mutation has been found to have a unique in vitro defect in T cell apoptosis associated with IL-2 withdrawal but normal T cell and B cell apoptosis in response to staurosporine, etoposide, cisplatin and radiation. This suggests the possibility of a distinct defect in the mitochondrial cell death pathway with a number of candidate proteins that could account for this in vitro defect. This lead to protein analysis by Western blot using B cell lines from the patient that revealed normal levels of Bim, Bad, phosphorylated-Bad, Bak, Bax and Bcl-2. In addition sequencing of genomic DNA revealed no evidence of a mutation in the gene encoding Bim and using cDNA, no evidence of a mutation in the gene encoding Bak. In light of the clearly demonstrated defect in apoptosis uniquely associated with cytokine withdrawal and the negative studies of candidate proteins, we have begun evaluation of this patients lymphocytes compared to normals using apoptosis-specific and whole human genome microarrays. At the same time we are further evaluating the nature of cytokine withdrawal induced cell death based on this patients since the in vitro functional findings suggest that this death pathway has distinct features that differ from other mitochondrial death pathways induced by drugs (etoposide, cisplatin) and irradiation. The Bim sequence is normal including over 600 bases upstream from exon 1 that includes the forkhead binding domain as well as sequencing demonstrating that the poly A site is normal. In light of the consistently lower levels of Bim including at baseline studies are currently underway to evaluate the function and sequence of the forkhead O proteins including Fox O1, Fox O3 and Fox O4. Additional work using microarray screening revealed that there was likely upregulation in the activity of NRAS based on the pattern increased mRNA expression. This lead to identification of a gain of function mutation in NRAS. Follow-up studies have identified that the siRNA knockdown of BIM in normal cells decreased sensitivity to cytokine induced apoptosis. In addition, transfection with mutant NRAS recapitulates the apoptotic defect in normal cells. The results of these expanded studies have been published in the PNAS.